Spatiotemporal resolution of T-box function in early mesoderm patterning : an optochemical approach
- Zebrafish mesoderm development is largely regulated by a network of T-box transcription factors comprised of the Brachyury ortholog no tail a (ntla), spadetail (spt), and tbx6-like (tbx6l). Conventional genetic mutant analyses have demonstrated the importance of these factors in mesoderm patterning; however, the non-conditionality of these perturbations have limited our understanding of how these genes function in space and time. Exploiting the optical transparency of early zebrafish embryos, our lab has developed photoactivatable caged morpholinos (cMO) that permit light-inducible gene silencing with spatiotemporal precision. The focus of this dissertation is on the application of these optochemical probes to resolve the function of the T-box gene spt in two distinct developmental contexts: 1) paraxial mesoderm patterning and 2) posterior medial floor plate (MFP) formation. In Chapter 1, I provide a brief survey of zebrafish mesoderm development, describe the dynamics of T-box gene expression during gastrulation, and detail the mesodermal phenotypes observed upon loss or gain of function of these factors. In Chapter 2, I apply a cMO targeting spt with photoactivatable lineage tracers, fluorescence-activated cell sorting, and RNA-sequencing to reveal 124 transcripts that are differentially expressed within a population of cells enriched for paraxial mesoderm progenitors upon loss of Spt function. Furthermore, I uncover a novel Spt-dependent regulation of collinear hox gene activation in these cells. In Chapter 3, I combine the spt cMO with a conventional ntla MO to characterize the temporal and spatial requirements of these genes in posterior MFP development. These studies indicate that ntla and spt act specifically within lateral margin derived progenitors during gastrulation to promote posterior morphogenetic movement of MFP progenitors in the midline without affecting Nodal-dependent MFP fate specification. This dissertation highlights the advantages of light-activated technologies to spatiotemporally characterize gene function during early vertebrate development.
|Type of resource
|electronic; electronic resource; remote
|1 online resource.
|Payumo, Alexander Y
|Stanford University, Department of Chemical and Systems Biology.
|Wysocka, Joanna, Ph. D
|Wysocka, Joanna, Ph. D
|Statement of responsibility
|Alexander Y. Payumo.
|Submitted to the Department of Chemical and Systems Biology.
|Thesis (Ph.D.)--Stanford University, 2015.
- © 2015 by Alexander Yap Payumo
- This work is licensed under a Creative Commons Attribution Non Commercial 3.0 Unported license (CC BY-NC).
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